The present invention is directed to the analysis of RNA molecules by liquid chromatography. More specifically, the invention is directed to a liquid chromatography system and method, such as Matched Ion Polynucleotide Chromatography, which enhances the purification of RNA.
RNA molecules are polymers comprising sub-units called ribonucleotides. The four ribonucleotides found in RNA comprise a common cyclic sugar, ribose, which is covalently bonded to any of the four bases, adenine (a purine), guanine (a purine), cytosine (a pyrimidine), and uracil (a pyrimidine), referred to herein as A, G, C, and U respectively. A variety of modified bases are also encountered in RNA. A phosphate group links a 3xe2x80x2-hydroxyl of one ribonucleotide with the 5xe2x80x2-hydroxyl of another ribonucleotide to form a polymeric chain. Secondary structure commonly occurs via intra-chain hydrogen bonds between complementary bases.
Ribonucleic acid (RNA) transports genetic information within the cell. In the most general terms, RNA passes specific peptide and protein coding information from the genome in the nucleus to those parts of the cell responsible for the production of these peptides and proteins. There are a number of different RNAs found in the cell at any given time. Some of them are noted below, along with their functions:
Messenger RNA (mRNA): Carries the coding messages to the ribosomes for the production of peptides and proteins;
Small nuclear RNA (snRNA): Responsible for removing intronic sequences from mRNA precursors (preribosomal mRNAs), prior to the spliced mRNA delivery to the ribosomes;
Transfer RNA (tRNA): Provide chemically activated amino acids for binding to the ribosomal complex in the process of protein synthesis;
Ribosomal RNA (rRNA): RNA within the ribosomes themselves, which by association, are part of the protein synthesis process.
Within molecular biology, it is often necessary to isolate these RNA molecules, particularly mRNA. This is because an mRNA xe2x80x9cmessagexe2x80x9d indicates that a gene has been transcribed. Furthermore, the extent to which the gene is expressed (up-regulated, down-regulated, turned on, turned off) is often proportional to the amount of gene-specific RNA (mRNA) present in the cell. The quantitation of gene expression via mRNA content can occur by various means (e.g., RT-PCR, expression array/hybridization analysis, etc.)
On other occasions it is desirable to create a compilation, or xe2x80x9clibraryxe2x80x9d, of those genetic messages being expressed in a cell or cells under a given set of conditions (i.e., normal vs. diseased state). This is often performed by selectively harvesting the mRNAs present in a sample, reverse transcribing the mRNAs to cDNA (first strands and second strands), and then cloning these double stranded sequences into some suitable vector. Once cloned, the cDNA xe2x80x9clibrariesxe2x80x9d can be utilized in various procedures.
RNA is thus the starting material in numerous molecular biology experiments involving the identification of unknown genes and assignment of functions to various proteins. Quality, quantity, purity, and size distribution of RNA determine the rate of success in applications such as cDNA library construction, Northern blot analysis, reverse transcription, and in situ analysis. Present techniques for the purification of RNA are labor intensive and lengthy. Quantification is routinely performed by gel visualization, radiometric methods, and spectrophotometric techniques. Sizing and quality determination is often performed by electrophoresis on denaturing agarose gels. However, RNA can become covalently modified by the chemicals used during the fractionation process (e.g., formaldehyde or acryalmide). Many of the present separation techniques require the use of hazardous chemicals (e.g., methylmercuric hydroxide, CsCl) such as described in Current Protocols in Molecular Biology, F. M. Ausubel et al. (1995) Eds., John Wiley and Sons, pp. 4.0.1-4.10.11 and in J. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, New York.
In the preparation of mRNA from total RNA, spin columns containing beads coated with poly T oligomers are often used (e.g., Poly(A)Pure(trademark) mRNA Purification Kit, Ambion, Inc., Austin, Tex.; Oligotex(trademark) mRNA Purification System, Qiagen, Inc., Valencia, Calif.). The disadvantages of this technique include a requirement for high amounts of total RNA sample due to low recovery of mRNA, contamination of the product (e.g. by rRNA), and degradation of the mRNA product.
There is a need for faster, safer, more reliable, less labor intensive, and more accurate methods of RNA analysis.
Separations of polynucleotides such as RNA have been traditionally performed using electrophoresis through agarose gels or sedimentation through sucrose gradients. However, liquid chromatographic analysis of polynucleotides is becoming more important because of the ability to automate the process and to collect fractions.
Traditional chromatography is a separation process based on partitioning of mixture components between a xe2x80x9cstationary phasexe2x80x9d and a xe2x80x9cmobile phasexe2x80x9d. The stationary phase is provided by the surface of solid materials which can comprise many different materials in the form of particles or passageway surfaces of cellulose, silica gel, coated silica gel, polymer beads, polysaccharides, and the like. These materials can be supported on solid surfaces such as on glass plates or packed in a column. The mobile phase can be a liquid or a gas in gas chromatography.
The separation principles are generally the same regardless of the materials used, the form of the materials, or the apparatus used. The different components of a mixture have different respective degrees of solubility in the stationary phase and in the mobile phase. Therefore, as the mobile phase flows over the stationary phase, there is an equilibrium in which the sample components are partitioned between the stationary phase and the mobile phase. As the mobile phase passes through the column, the equilibrium is constantly shifted in favor of the mobile phase. This occurs because the equilibrium mixture, at any time, sees fresh mobile phase and partitions into the fresh mobile phase. As the mobile phase is carried down the column, the mobile phase sees fresh stationary phase and partitions into the stationary phase. Eventually, at the end of the column, there is no more stationary phase and the sample simply leaves the column in the mobile phase.
A separation of a mixture of components occurs because the mixture components have slightly different affinities for the stationary phase and/or solubilities in the mobile phase, and therefore have different partition equilibrium values. Therefore, the mixture components pass down the column at different rates.
In traditional liquid chromatography, a glass column is packed with stationary phase particles and mobile phase passes through the column, pulled only by gravity. However, when smaller stationary phase particles are used in the column, the pull of gravity alone is insufficient to cause the mobile phase to flow through the column. Instead, pressure must be applied. However, glass columns can only withstand about 200 psi. Passing a mobile phase through a column packed with 5 micron particles requires a pressure of about 2000 psi or more to be applied to the column. 5 to 10 micron particles are standard today. Particles smaller than 5 microns are used for especially difficult separations or certain special cases). This process is denoted by the term xe2x80x9chigh pressure liquid chromatographyxe2x80x9d or HPLC.
HPLC has enabled the use of a far greater variety of types of particles used to separate a greater variety of chemical structures than was possible with large particle gravity columns. The separation principle, however, is still the same.
An HPLC-based ion pairing chromatographic method was recently introduced to effectively separate mixtures of polynucleotides wherein the separations are based on base pair length (U.S. Pat. No. 5,585,236 to Bonn (1996); Huber, et al., Chromatographia 37:653 (1993); Huber, et al., Anal. Biochem. 212:351 (1993)). Ion pair reverse phase high pressure liquid chromatography (IPRPHPLC) was used as a process for separating DNA using non-polar separation media, wherein the process used a counterion agent, and an organic solvent to release the DNA from the separation media. This method was used in the separation of double stranded and single stranded DNA.
Objects of the present invention include providing a method and system for segregating RNA molecules which is fast, safe, reliable, convenient, reproducible, and quantitative.
In one aspect, the invention provides a chromatographic method for segregating a mixture of RNA molecules. The method includes applying the mixture to a polymeric separation medium having non-polar surfaces, and eluting said RNA molecules with a mobile phase which includes counterion reagent and an organic component. The preferred surfaces are characterized by being substantially free from multivalent cations which are free to interfere with RNA segregation. The elution of the RNA molecules is preferably carried out at a minimum denaturing temperature. For example, the elution can be carried out at a temperature greater than about 60xc2x0 C. Preferably, the elution is carried out at a temperature within the range of about 60xc2x0 C. to about 100xc2x0 C. The pH of the mobile phase is preferably within the range of about pH 5 to about pH 9. The method is useful in the segregation of RNA molecules exceeding about 1,000 nucleotides and can be used in the segregation of RNA molecules having up to about 20,000 nucleotides.
In the method, the segregation can be performed using a column containing polymeric beads having non-polar surfaces, wherein the beads have an average diameter of about 1 to about 100 microns. The separation medium can include polymer beads having an average diameter of 0.5 to 100 microns, the non-polar surfaces being unsubstituted or having bound thereto a hydrocarbon group having from 1 to 1,000,000 carbons.
The separation can be performed using a column including a polymeric monolith. The surfaces of the monolith can be unsubstituted or substituted with a hydrocarbon group having from 1 to 1,000,000 carbons. The monolith can include monovinyl substituted aromatic compound, divinyl substituted aromatic compound, acrylate, methacrylate, polyolefin, polyester, polyurethane, polyamide, polycarbonate, fluoro-substituted ethylene, or combinations of one or more thereof.
The method can include collecting mobile phase fractions containing the RNA molecules. The method can include detecting RNA molecules (e.g. using UV detection) during the elution step. The segregating is preferably performed by Matched Ion Polynucleotide Chromatography. The mobile phase preferably includes a counterion agent and an organic solvent, wherein the organic solvent is water soluble.
Examples of multivalent metal cations that can interfere with the segregation include Fe(III), Cu(II), Cr(III), and colloidal metal. During the elution, the mobile phase can include a multivalent cation binding agent such as EDTA.
Examples of an organic solvent useful in the method include alcohol, acetonitrile, dimethylformamide, tetrahydrofuran, ester, ether, or mixtures of one or more thereof. In the method, the counterion agent can be selected from lower alkyl primary amine, lower alkyl secondary amine, lower alkyl tertiary amine, lower trialkyammonium salt, quaternary ammonium salt, or mixtures of one or more thereof. Non-limiting examples of preferred counterion agents include octylammonium acetate, octadimethylammonium acetate, decylammonium acetate, octadecylammonium acetate, pyridiniumammonium acetate, cyclohexylammonium acetate, diethylammonium acetate, propylethylammonium acetate, propyldiethylammonium acetate, butylethylammonium acetate, methylhexylammonium acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethydiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, triethylammonium hexafluoroisopropyl alcohol, or mixtures of one or more thereof.
In the method, the separation medium can be subjected to a prior acid wash treatment to remove any residual metal contaminants. In the method, the separation medium can be subjected to a prior treatment with multivalent cation binding agent.
In another aspect, the invention provides a chromatographic method for segregating a mixture of RNA molecules having lengths exceeding about 100 nucleotides. The method includes (a) flowing the mixture through a separation column containing polymer beads having an average diameter of 0.5 to 100 microns, the beads being unsubstituted polymer beads or polymer beads substituted with a moiety selected from the group consisting of hydrocarbon having from 1 to 1,000,000 carbons, and (b) segregating the mixture of RNA molecules at a minimum denaturing temperature.
In yet another aspect, the invention provides a chromatographic method for segregating a mixture of RNA molecules. The method includes (a) flowing the mixture through a separation column containing polymer beads having an average diameter of 0.5 to 100 microns, the beads being unsubstituted polymer beads or polymer beads substituted with a moiety selected from the group consisting of hydrocarbon having from 1 to 1,000,000 carbons. The preferred beads are characterized by being substantially free from multivalent cations which are free to bind with said RNA, (b) segregating said mixture of RNA molecules at a minimum denaturing temperature.
In a further aspect, the invention includes an improved method for segregating a mixture of RNA molecules by Matched Ion Polynucleotide Chromatography, said mixture comprising molecules having lengths exceeding about 100 nucleotides. The method includes (a) applying a solution of the molecules and counterion reagent to a column containing polymeric separation beads having non-polar surfaces, wherein the separation beads have an average diameter of 1 to 100 microns, the column having an ID greater than about 5 mm and (b) eluting the RNA molecules with a mobile phase which includes the counterion reagent and an organic component. The method can be carried out at a temperature within the range of about 60xc2x0 C. to about 90xc2x0 C. The ID can be greater than about 7 mm. The ID can be greater than about 10 mm. The ID is preferably within the range of about 5 mm to about 1 m.
In still another aspect, the instant invention concerns an improved reverse phase chromatography column for segregating a mixture of RNA molecules by Matched Ion Polynucleotide Chromatography, the mixture comprising molecules having lengths exceeding about 100 nucleotides. The improved column includes a cylinder containing polymer beads, the beads having an average diameter of 1 to 100 microns, the beads being unsubstituted polymer beads or polymer beads substituted with a hydrocarbon moiety having from 1 to 1,000,000 carbons, the column having an ID greater than about 5 mm.
In a yet further aspect, the invention concerns a chromatography system for segregating a mixture of RNA molecules by Matched Ion Polynucleotide Chromatography which includes a cylindrical column containing polymer beads, the beads having an average diameter of 1 to 100 microns, the beads being unsubstituted polymer beads or polymer beads substituted with a hydrocarbon moiety having from 1 to 1,000,000 carbons, the column having an ID greater than about 5 mm.
In a further aspect, the invention provides a chromatographic method for segregating a mixture of RNA molecules having lengths exceeding about 100 nucleotides. The method includes (a) applying a solution of the fragments and counterion reagent to a column containing polymeric beads having non-polar surfaces, wherein the beads have an average diameter of about 1 to about 100 microns, wherein the surfaces are characterized by being substantially free from multivalent cations which are free to bind with the RNA and (b) eluting the RNA molecules with a mobile phase which includes said counterion reagent and an organic component. The eluting can be carried out at a minimum denaturing temperature.